1. Field of the Invention
The present invention relates to the determination of the presence of leukocytes, or other esterase activity, in a test sample. Moreover, the invention relates to a semi-quantitative test which is rapid and which dispenses with the need for microscopic examination or wet chemistry laboratory procedures.
The presence of an abnormal level of leukocytes in a patient's urine is possibly indicative of such pathological conditions as kidney or urogenital tract infection or other dysfunction. Accordingly, accurate urinary leukocyte information can be an invaluable tool to the physician in diagnosis and treatment of such pathologies. Traditionally, the medical profession has relied on visual determination techniques to count leukocyte population in urine sediment or uncentrifuged urine, a process requiring expensive equipment such as a centrifuge and microscope, as well as a great deal of time on the part of the clinician. Moreover, the traditional techniques suffer from the inadequacy that only intact cells are determined. Leukocytes occurring in the urinary system are subject to conditions which can favor extensive cell lysis. For example, it is known that in urines of abnormally high pH, leukocyte half life can be as low as 60 minutes. Since lysed cells escape detection in visual examination techniques, erroneously low determinations and false negatives can result.
Of the two techniques of microscopic leukocyte analysis--urine sediment and non-centrifuged, homogenized urine--the former is clearly the most desirable. Although dependable results can inure to the latter, urine sediment observation is used in an overwhelming majority of instances. It requires that the urine sample be centrifuged and the sediment isolated and subjected to microscopic inspection. The analyst then counts the number of leukocytes appearing in the viewing field. However, this task is further complicated by the presence of other urinary components in the sediment such as epithelial cells and salt particles. The varying content of sediment constituents, coupled with other complicating factors including non-homogeneity of the sample and varying optical powers of microscope equipment, can lead to enormous errors in the ultimate determination.
It is thus apparent that a quick, facile method of leukocyte determination, one which would eliminate the need for time-consuming techniques, as well as cost-consuming equipment, and which would provide accurate results whether the cells were intact or had been lysed, would indeed advance the state of the art by a quantum jump. The present invention provides such an advance. It is based, not on the ability to see leukocytes, but on the enzymatic activity they exhibit, and therefore is not subject to the inaccuracies described above.
2. Description of the Prior Art
There exists in the prior art a body of references which disclose the use of certain esters which, when cleaved by enzymatic activity, result in the formation of color or other detectable species. Thus, British Patent No. 1,128,371 discloses the use of indoxyl and thioindoxyl esters as useful chromogens in detecting hydrolytic enzymes in body fluids. The enzymes cleave the ester to generate free indoxyl, which subsequently oxidizes to form the dimeric product indigo, a readily observable blue dye. Such activity is said to be due to, among other enzymes, cholinesterase. This patent also teaches that, in addition to the indoxyl portion of the ester substrate, the acid radical is chosen with particular reference to the enzyme to be detected. For example, it is stated that the acid radical can be acetate, laurate or stearate for detection of esterase or lipase. For detecting enzymes such as phosphatase or sulfatase the acyl radical can be inorganic. Thus, the British Patent can be held to teach the use of chromogenic esters as substrates for determining esterolytic enzymes, such esters comprising indoxyl or thioindoxyl as the alcoholic moiety of the ester, the acyl moiety being tailored to be conducive to the particular enzyme to be determined.
The effect of careful acyl radical selection is nowhere more clearly exemplified than in two references which demonstrate esterase specificity for esters in which the acyl radical comprises an N-protected amino acid or peptide. Thus Janoff, et al., Proc. Soc. Exper. Biol. Med. 136:1045-1049 (1971) teaches that alanine esters are specific substrates for esterase obtained from human leukocytes. Specifically this reference teaches that an extract of human leukocyte granules hydrolyzed n-acetyl-l-alanyl-l-alanyl-l-alanine methyl ester. Moreover, l-alanine-p-nitrophenol ester was similarly hydrolyzed to yield the yellow p-nitrophenol colorform.
Similarly, Sweetman et al., Jour. Hist. Soc., 22:327-339 teaches the use of 1-naphthyl N-acetyl-DL-alanine and 1-naphthyl butyrate to demonstrate the presence of esterase, as well as 1-naphthyl N-acetyl-l-alanyl-l-alanyl-1-alanine.
U.S. Pat. No. 4,278,763, assigned to Boehringer Mannheim GmbH combines these teachings in arriving at the indoxyl or thioindoxyl esters of amino acids or peptides as still another example of a traditional colorogenic substrate for leukocytic esterase activity. Like the Janoff and Sweetman references, the Boehringer patent teaches the equivalence of proteases and esterase in their esterolytic penchants.
Other prior art teachings with respect to leukocyte determination include the peroxidative activity of granular leukocytes (U.S. Pat. No. 3,087,794). Such an approach, however cannot distinguish between leukocytes and hemoglobin, and one cannot tell whether a positive test is indicative of white or red blood cells.
Finally, there is available commercially a product known as "Cyturtest" which utilizes N-tosylalanine indoxyl ester referred to, supra. This product comprises a filter paper pad impregnated with the amino acid indoxyl ester. The pad is mounted on a plastic strip. When immersed in a leukocyte-containing urine sample, a color appears on the filter paper pad due to the formation of indigo. However, this test suffers from the drawback of having a considerably lengthy waiting period (about 15 minutes) before the test results can be assessed.
Thus, although the use of chromogenic esterase substrates is well-entrenched in the prior art, and despite the existance of esterase affinity towards indoxyl alaninate and peptidate, there exists no truly rapid test for leukocytes in urine. It is towards solving this problem that the present invention is directed.
Based on a concerted effort of research and development, it was discovered that a particularly facile test for the presence of leukocytes in urine could be had in a test time of only 3-4 minutes and less, thus overcoming a serious drawback of the prior art.